Recovery and evaluation of somatic cells from ovine and bovine semen for use in nuclear transfer /
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| Other Authors: | , |
| Format: | Thesis eBook |
| Language: | English |
| Published: |
[College Station, Tex.] :
[Texas A&M University],
[2010]
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| Subjects: | |
| Online Access: | Link to OAK Trust copy |
| Abstract: | Somatic cells in semen are a potential source of nuclei for cloning animals by somatic cell nuclear transfer. Culture of the cells from frozen semen, if possible, would be extremely valuable for preservation or restoration of endangered, exotic, and extinct animals when other ways of obtaining somatic cells are unavailable. In the present study, somatic cells isolated from ovine and bovine semen samples were characterized, culture systems were evaluated for attachment and proliferation of these cells, and usefulness of these cells for somatic cell nuclear transfer was determined. Semen samples were collected from eight rams representing three breeds: Dorper, Suffolk, and Hampshire and nine bulls representing three breeds: Charolais, Brahman, and a crossbred Brahman. Somatic cells were isolated immediately post collection by centrifuging through percoll columns and the epithelial cells were identified by immunofluorescence analysis. Culture systems were evaluated for their ability to support attachment and proliferation of the cells. A supplemented medium composed of DMEM/F12, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 30 ug/ml bovine pituitary extract, 5 ug/ml insulin, 10 ng/ml cholera toxin, and 50 ug/ml gentamicin significantly improved cell proliferation over sheep fetal fibroblastconditioned medium, 3T3 cell-conditioned medium, and basic medium (p<0.05). Cell proliferation and attachment were further improved when Matrigel-coated culture surfaces were used (p<0.05). However, the system was not adequate for obtaining cell growth from frozen semen. To check the chromosome anomalies, metaphase chromosomal complements of the cells cultured from 4 rams were evaluated. The predominant chromosome number of cells from three of the rams (Dorper 18-month-old; Suffolk 17-month-old; Suffolk 18-month-old) was 2n = 54, which is the normal modal number for sheep. However, the numbers of chromosomes of cells cultured from the fourth ram (Hampshire, 18-month-old)were near-triploid. These results indicate the need for chromosome analysis of cells before using them for cloning experiments. In our attempts to clone animals, blastocyst stage embryos were successfully produced using epithelial cells cultured from semen of three different bulls. However, no compact morulae or blastocysts were obtained when somatic cells isolated from frozen semen but not cultured were used as donor cells. |
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| Item Description: | "Major Subject: Veterinary Physiology" Title from author supplied metadata (automated record created 2010-03-12 12:08:51). Electronic resource. |
| Physical Description: | 1 online resource. |
| Bibliography: | Includes bibliographical references. |